A series of coordinating events must occur for an intracellular bacteria to gain entry into and establish infection within its host cell. This application is directed at understanding the molecular signals required for the bacteria species, Legionella pneumophila, to infect a human macrophage cell. L. pneumophila is the etiological agent of Legionnaire's disease, a serious type of human pneumonia. The natural reservoir of L. pneumophila is the amoeba, but infection also occurs in humans when the bacterium invades alveolar macrophages. Previous work has determined that transient phosphorylation of host cell extracellular signal regulated kinase (ERK) occurs shortly after exposure to L. pneumophila. The importance of this event in the bacterial lifecycle is unknown, but in Dictyostelium discoideum, an amoebal species that supports L. pneumophila growth, regulation of ERK activity is extremely important. The role of dual specific phosphatases (DUSPs) in human infection is also unclear, however, DUSP 1, 2, and 6, all of which negatively regulate MAP kinases, are highly upregulated at the transcriptional level in a human cell line challenged with L. pneumophila. From these observations we predict that tight control of ERK activation is critical for efficient intracellular replication of the microorganism. To test this hypothesis three integrated specific aims are proposed. Aim 1 will determine if the ERK signaling cascade is required for L. pneumophila replication, invasion, and host cell survival using siRNA knockdown, kinase assays, and pharmacological inhibitors. Aim 2 will clarify the role of the DUSP family of MAPK phosphatases in L. pneumophila infection using gene silencing and microarray profiling. Aim 3 will define whether feedback mechanisms exist between ERK and DUSP. Immunoblotting, real-time RT-PCR, and protein localization experiments using confocal microscopy in cells with siRNA knockdown of either ERK or DUSP will determine if these proteins control each other's function and intracellular localization. Together this work will provide insight into how ERK activity is controlled, which will contribute to our understanding of both basic cellular signaling processes and also pathogenesis of human bacterial infections. PUBLIC HEALTH RELEVANCE: This application is directed at identifying novel host cell factors that are critical in supporting L. pneumophila infection. As 8,000-25,000 individuals in the United States develop Legionnaire's disease each year and the fatality rate of this pneumonia is over 20 percent, understanding the molecular mechanisms that control infection will aid in the advancement of medical care and the development of new drug therapies.